Identification of a new Ehrlichia species from a patient suffering from Ehrlichiosis

ABSTRACT

A new isolate of Ehrlichia species has been obtained from a patient suffering from ehrlichiosis. The new isolate has been found to be similar, but distinctly different from E. canis. A diagnostic kit and methods for diagnosing ehrlichiosis in humans and for screening drugs toxic to the new isolate have been described.

This application is a continuation of application Ser. No. 08/394,464,filed Feb. 27, 1995, abandoned, which is a Divisional of applicationSer. No. 08/147,891, filed Nov. 5, 1993, now U.S. Pat. No. 5,413,931,which is a Continuation of 07/687,526, filed Apr. 18, 1991 abandoned

This invention relates to the identification and characterization of anew microorganism isolated from a patient suffering from ehrlichiosis.The new organism, designated herein as Ehrlichia chaffeensis, is similarto but distinct from Ehrlichia canis.

BACKGROUND OF THE INVENTION

Human ehrlichiosis is a newly recognized disease characterized by fever,headache, malaise, thrombocytopenia, leukopenia, and elevated liverenzymes (Anon., M.M.W.R. 37, 270, 275, 1988; Fishbein, et al., JAMA 257,3100, 1987; Fishbein, et al., J. Infect. Dis. 160, 803,1989; Eng, etal., JAMA 264, 2251, 1990). Often the patients also have a history oftick exposure. The only Ehrlichia species known to infect humans isEhrlichia sennetsu, the agent responsible for sennetsu rickettsiosis, adisease that has been reported only in Japan and Malaysia (Ristic, inMicrobiology 1986, L. Leive, Ed., American Society for Microbiology,Washington, D.C., 1986, pp. 182-187). Since recognition of a human formof ehrlichiosis in the United States in 1986, laboratory-basedsurveillance has led to the identification of about 215 persons withvariable antibody titer to E. canis in 20 states, predominantly insoutheastern and south central areas of the United States (Fishbein, etal., J. Infect. Dis., 160, 803, 1989; Eng, et al., JAMA 264, 2251,1990). It may be noted, however, that despite such serologic evidence,the causative agent of human ehrlichiosis remained unidentified and theetiology of the disease also remained undetermined.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to isolate,identify and characterize the agent associated with human ehrlichiosis,the agent thus isolated having been designated herein as "Ehrlichiachaffeensis" or "human Ehrlichia".

It is noted that if the scientific community accepts the change ofnomenclature of E. chaffeensis to E. homosapiensis or other designation,then of course it should be recognized accordingly.

It is another object of the present invention to grow the Ehrlichiachaffeensis isolate in a cell culture.

It is also an object of the present invention to provide a recombinantmolecule or construct containing E. chaffeensis nucleotide sequence orE. chaffeensis-specific fragment thereof.

A further object of the present invention is to prepare antibodieshaving specificity particularly against E. chaffeensis.

A still further object of the present invention is to provide clonedgenes of E. chaffeensis that encode E. chaffeensis-specific antigens.

An additional object of the present invention is to provide acomposition comprising an immunogenic amount of E. chaffeensis antigen,either naturally produced or recombinantly made, to induce antibodiesagainst E. chaffeensis in a host susceptible to infection by E.chaffeensis.

A further object of the present invention is to provide an immunoassayfor detecting human ehrlichiosis employing E. chaffeensis or a fragmentderived therefrom as an antigen.

Another object of the present invention is to provide a diagnostic kitcomprising a container containing E. chaffeensis-specific antigen orantibody.

Yet another object of the present invention is to provide a method forscreening the toxicity of a drug against E. chaffeensis by comparing thegrowth of E. chaffeensis in the presence and absence of the drug in acell culture environment.

Various other objects and advantages will become evident from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects, features and many of the attendantadvantages of the invention will be better understood upon a reading ofthe following detailed description when considered in connection withthe accompanying drawing wherein:

FIG. 1 shows transmission electron micrograph of the human Ehrlichiaisolate in the cytoplasm of a DH82 cell. Cell cultures were scraped fromflasks and centrifuged at 180 X g for 10 minutes. The resulting pelletswere fixed at 4° C. in 2.5% 0.2M phosphate buffered glutaraldehyde,post-fixed in 1% buffered osmium tetroxide, dehydrated in a standardethanol series, and embedded in a modified Araldite-Epon mixture.Sections were stained with uranyl acetate and lead citrate. Organisms(arrows) are seen in a membrane-bound morulae. Bar=0.5 μm.

FIG. 2 shows the 16S rRNA nucleotide sequences of E. chaffeensis (SEQ IDNO: 1) and E. canis (SEQ ID NO: 2).

DETAILED DESCRIPTION OF INVENTION

The above and various other objects and advantages of the presentinvention are achieved by obtaining a biologically pure isolate ofEhrlichia chaffeensis, its cloned genes and antigenic products.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Isolation, Identification and Characterization of E. chaffeensis

A 21-year-old man (Table 1, patient no. 1) was admitted to a medicalclinic in Arkansas on Jul. 19, 1990 with fever (103° F.), headache,nausea, and vomiting. A physical examination revealed prominent cervicallymphadenopathy, splenomegaly and no rash. Multiple excoriated lesionsfrom constant (11 days) exposure to ticks, chiggers, and mosquitoes wereobserved. Five days after the onset of illness, hematocrit was 40.1%,the white cell count was 2200 per cubic millimeter, and the plateletcount was 100,000 per cubic millimeter.

A small volume of blood (30 ml. heparin and 5 ml. EDTA) was drawn fromthe patient and shipped with cold packs to the Centers for DiseaseControl. The leukocytes were separated from the red blood cells (30 ml.heparinized whole blood) approximately 24 hours after collection, andlayered onto a previously established monolayer of DH82 (continuouscanine macrophage) cells with minimum essential medium supplemented with1% L-glutamine and 12.5% heat-inactivated fetal bovine serum (Dawson, etal., J. Infect. Dis., 163, 564, 1991). The culture was maintained at 37°C. and monitored by direct immunofluorescence using a fluoresceinconjugate prepared from the serum of a patient with ehrlichiosis.

Organisms closely resembling ehrlichieae were first observed in thecytoplasm of cultured macrophages 35 days after the addition of theinfected blood (FIG. 1). Thereafter, the proportion of infectedmacrophages increased, reaching a maximum of 80% on day 48. Uninoculatedcontrol cultures of the DH82 cells remained free of organisms.

Electron microscopic examination of the infected cells revealed thatinclusion bodies were surrounded by a distinct cytoplasmic membrane(FIG. 1). Each individual organism was surrounded by two membranes, theinner plasma membrane and the outer cell wall. The organisms were alsoextremely pleomorphic, ranging in shape from oval to boomerang todiamond.

The human isolate thus obtained appears to be antigenically related tothe etiologic agent of human ehrlichiosis as suggested by the positiveindirect immunofluorescence reactions obtained when serum samples wereexamined from 12 patients previously diagnosed by the indirectimmunofluorescent antibody test, and 2 patients suspected of havingehrlichiosis based on the clinical symptoms (Table 1). Serum specimensfrom the 14 patients reacted strongly with the newly isolated organism.In two cases (patients no. 1 and 6), a specific fluorescein response wasobserved only with the human isolate. The negative control sera, fromhealthy adults, showed no reaction to either organism.

DNA was extracted from the original whole blood sample (EDTA) andutilized as a polymerase chain reaction (PCR) template to produceamplified DNA for cloning and sequencing. DNA was also extracted fromthe DH82 cell line infected with the new isolate, with E. canis Oklahomaisolate as described by Dawson, et al., J. Infect. Dis. (163, 564,1991), and uninfected DH82, for similar amplification and sequencecomparison. Samples were amplified for 40 cycles in a thermal cyclerusing degenerate primers specific for the 3' half of eubacterial 16Sribosomal RNA (rRNA) (Wilson, et al., J. Clin. Microbiol. 28, 1942,1990) and containing unique restriction sites on the 5' ends. PCRproducts corresponding to the 16S rRNA sequence were seen in all samplesexcept when uninfected DH82 derived DNA was used as a template. Theresulting PCR products were cloned into pUC19 and sequenced. All sampleswere amplified, cloned and sequenced independently 2 times to preventthe reading of Taq polymerase incorporation errors. The PCR product fromthe patient's blood sample matched the product from the new isolategrown in the DH82 cells for all 683 nucleotides defined within the PCRprimers. A comparison with available sequence data also revealed that itwas 86.8% related to E. risticii (Genbank Accession # M21290), arecently isolated equine pathogen. Serologic data and 16S rRNAsequencing further indicated that the newly isolated Ehrlichia issimilar, but not identical to E. canis. FIG. 2 shows the comparativenucleotide sequences of the 16S rRNA of E. canis and E. chaffeensis. Theassociation of the new isolate with human ehrlichiosis further indicatesthat the new isolate may be involved in the etiology of humanehrlichiosis.

Of course, the availability of the new Ehrlichia isolate of the presentinvention now makes it possible to prepare a composition comprising aneffective amount of Ehrlichia chaffeensis antigen to induce an immuneresponse to Ehrlichia chaffeensis in a host susceptible to infection byEhrlichia chaffeensis, and a pharmaceutically acceptable carrier. Adiagnostic kit in accordance with the present invention comprises atleast a container containing an antigen which reacts specifically withanti-Ehrlichia chaffeensis antibodies, and instructional material toperform the diagnostic test.

Similarly, a method for diagnosing human ehrlichiosis comprises the stepof reacting a sample of the biological fluid (such as blood, serumplasma and the like) or a tissue obtained from an individual suspectedof affliction with ehrlichiosis, with an E. chaffeensis specificantigen, the occurrence of a positive immunological reaction beingindicative of ehrlichiosis in said individual. An example of such adiagnostic test is the indirect fluorescent antibody (IFA) test asdescribed herein above. In order to prepare antigen slides for the IFAtest, cells from E. chaffeensis-infected DH82 cultures (80-90%infection) were suspended in culture supernatant. This suspension wasthen either used immediately or lyophilized and when necessaryreconstituted in distilled water. One drop (about 3 microliters) of theantigen was then placed onto each well of a teflon-coated slide. Theslides were air-dried for about 1 hour and stored at -90° C. As needed,slides were thawed and then fixed in acetone for about 15 minutes. Theserum sample was screened at a dilution of 1:64 in phosphate-bufferedsaline solution. When distinct staining of E. chaffeensis organisms wasobserved at this titer, serial two-fold dilutions were made. Serologicresults were recorded as the reciprocal of the highest dilution at whichspecific fluorescence of E. chaffeensis morulae were observed.

For the preparation of E. chaffeensis specific antibodies, Ehrlichiachaffeensis is grown in the DH82 cell line, or in any other cell linewhich will support the growth of E. chaffeensis, and purified by douncehomogenization followed by low speed centrifugation. Mice are theninoculated with this homogenate or any portion thereof. Afterapproximately 4 weeks, a couple of days before hybridoma formation, themice are given a booster inoculation. Spleens from primed and boostedmice are then harvested. Hybridomas are produced by fusion with anonsecretor mouse myeloma cell line (SP2/0) by the method of Kearney etal. (Kearney, et al, 1979, J. Immunol. 123:1548-1550). Selectedantibody-producing cultures identified by the IFA test or ELISA areexpanded in cell culture and stored frozen until cloning. Cells shown bythe IFA test or ELISA to be producing antibody to E. chaffeensis, areexpanded in cell culture from the frozen state and cloned by limitingdilution. The resulting monoclonal antibody-producing cultures are inturn expanded in cell culture. Selected clones are subsequentlyinoculated into mice for specific antibody production in ascitic fluids.These ascitic fluids are stored frozen until tested. Culture fluids andascitic fluids are evaluated by IFA, ELISA or any suitable immunoassay.

The availability of E. chaffeensis specific antibodies now makes itpossible to provide a diagnostic kit for detecting the presence of E.chaffeensis or E. chaffeensis antigens. Such a kit comprises at least acontainer containing an antibody which reacts specifically with E.chaffeensis antigens and instructional material to perform animmunoassay.

In order to determine the optimal treatment for human ehrlichiosis, thein vitro susceptibility to a candidate drug or a number of commonly usedantibiotics is determined. Approximately 10⁴ DH82 cells (at least 50% ofthese cells are infected with E. chaffeensis) are added to each well ofa 96 well microtiter plate. After a one hour incubation, the media isreplaced with media containing varying concentrations of tetracycline,doxycycline, minocycline, penicillin, erythromycin, gentamicin,rifampin, co-trimoxasole, ciprofloxacin or other drug of interest. Thepercentage of infected cells is then evaluated by wright giemsa stainand IFA daily for 8 days, the lesser the percentage of infected cells,the greater the toxicity of the drug.

Cloning and Purification of E. chaffeensis Antigens

E. chaffeensis is grown in the DH82 cell line or in any other cell linewhich will allow the growth of E. chaffeensis, and purified by standardrenograffin density gradient centrifugation. The Ehrlichia is then lysedand the DNA extracted via standard procedure using 1.0% SDS andproteinase K. The resulting DNA is then physically size fractionatedusing sonication and gel purification and linked with EcoRI linkers andcloned into lambda phage vector lambda zapII following standardprocedures such as described in Maniatis et al, 1982, MolecularCloning--A Laboratory Manual, Cold Spring Harbor, N.Y. The recombinantplaques are screened for antigen production via ELISA with primaryantibody being human convalescent sera absorbed with an E. coli lysate.Antigen expressing clones are subcloned.

Those subclones expressing E. chaffeensis specific antigens aresequenced and corresponding synthetic peptides are constructed from thededuced amino acid sequence for use as diagnostic antigens orimmunogens. Alternatively, recombinant antigens could be purified byaffinity chromatography or High Pressure Liquid Chromatography (HPLC)and the like.

Detection of Ehrlichiosis in Humans

Primers specific for E. chaffeensis have been constructed from the 16SrRNA sequence. The sequence of these primers is HE1=5'CAATTGCTTATAACCTTTTGGTTATAAAT 3' (SEQ ID NO: 3)HE3=5'TATAGGTACCGTCATTATCTTCCCTAT 3' (SEQ ID NO: 4). These primersdefine a 389 base pair product upon amplification and are also usefulfor amplifying DNA from organisms found in blood from patients withehrlichiosis using the standard polymerase chain reaction technique.Blood is processed similar to the amplification method used for RockyMountain spotted fever diagnosis (Tzianabos et al., J. Clin. Microbiol.,27:3866-2868, 1989. ) The resulting DNA is amplified for 40 cycles usinga thermal cycler. The correct size PCR-product is considered presumptiveevidence of ehrlichiosis. Test results indicate success with 4 out of 5infected human blood samples which were identified positive and 3uninfected blood samples which were identified negative. Anoligonucleotide probe may then be used to confirm the polymerase chainreaction product as belonging to the genus Ehrlichia. Such a probesequence may be 5' GCCATTAGAAATGATGGGTAATACTGTATAA 3'(SEQ ID NO: 5 ).

A second method for diagnosis would be to use the whole cell antigen orpurified E. chaffeensis as an ELISA antigen by solubilizing wholeEhrlichia and attaching to an ELISA plate. Human serum antibodies arethen allowed to react with this antigen and secondary anti-humanperoxidase-conjugated antibody is then reacted with the antigen-primaryantibody complex. The levels of human anti-Ehrlichia antibodies could bequantitated by reacting the complex with a colorimetric substrate forperoxidase or by other suitable method well known to one of ordinaryskill in the art.

DEPOSIT

A deposit of the DH82TIED (Human) cells infected with Ehrlichiachaffeensis in accordance with the present invention has been made underBudapest Treaty at the ATCC, Rockville, Md. on Jan. 29, 1991 underaccession number CRL 10679. The deposit shall be viably maintained,replacing if it becomes non-viable during the life of the patent, for aperiod of 30 years from the date of the deposit, or for 5 years from thelast date of request for a sample of the deposit, whichever is longer,and upon issuance of the patent made available to the public withoutrestriction in accordance with the provisions of the law. TheCommissioner of Patents and Trademarks, upon request, shall have accessto the deposit.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

                  TABLE 1                                                         ______________________________________                                        Table 1. Indirect fluorescent antibody titers of acute and convalescent       sera from 14 patients, tested with E. canis and the human Ehrlichia           isolate.                                                                      Starting at a dilution of 1:64, serial twofold dilutions of the acute         and                                                                           convalescent-phase sera were made in 0.15M PBS solution.                      Flourescein-conjugated rabbit anti-human IgG was prepared at the              Centers for Disease Control.                                                  Serologic results were reported as the reciprocal of the highest              dilution at which specific fluorescence of Ehrlichia morulae                  or individual organisms was observed.                                                  Tick     Days              Human                                     Patient  Exposure After             Ehrlichia                                 No.      State    Onset     E. canis                                                                              Isolate                                   ______________________________________                                        1*       AR        3        <64     <64                                                         39        <64     256                                       2        GA       11        <64     <64                                                         51        128     256                                       3        OK       26        <64     <64                                                         40        512     256                                       4        NJ        7        1024    1024                                                        25        512     512                                                         135       256     256                                       5        NC        8        <64     <64                                                         17        4096    2048                                      6        SC        8        <64     <64                                                         20        <64     2048                                                        28        <64     2048                                      7        WY       10        64      32                                                          24        512     512                                       8        TX       16        4096    4096                                                        28        1028    1028                                      9        MO       13        <64     <64                                                         24        512     1028                                      10       AR       10        512     512                                                         24        16384   32768                                     11       TN        -19**    <64     <64                                                         55        256     256                                       12       VA       18        32768   32768                                                       38        8192    8192                                      13       FL       10        1024    2048                                                        28        8192    16384                                     14       OK        8        <64     <64                                                         21        4096    16384                                     ______________________________________                                         * = patient from whom Ehrlichia isolate was made                              ** = specimen obtained 19 days before onset of disease                   

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 683 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACGCTGTAAACGATGAGTGCTAAATGTGAGGATTTTATCTTTGTATTGTAGCTAACGCGT60                TAAGCACTCCGCCTCCCCACTCAGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGAC120               CCGCACAAGGCTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTTTT180               TGACATGAAGGTCGTATCCCTCCTAATAGGGGGAGTCAGTTCGGCTGGACCTTACACAGG240               TGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG300               CAACCCTCATCCTTAGTTACCAACAGGTAATGCTGGGCACTCTAAGGAAACTGCCAGTGA360               TAAACTGGAGGAAGGTCCCCATGATGTCAAGTCAGCACGGCCCTTATAAGGTGGGCTACA420               CACGTGCTACAATGGCAACTACAATAGGTCGCGAGACCGCAAGGTTTAGCTAATCCATAA480               AAGTTGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGTCGGAATCGCTAGT540               AATCGTGGATCATCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCA600               CGCCATGGGAATTGGCTTAACTCGAAGCTGGTGTGCTAACCGCAAGGAAGCAGCCATTTA660               AGGTTGGGTTAGTGACTAGGGTG683                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 683 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ACGCTGTAAACGATGAGTGCTAAATGTGAGGATTTTATCTTTGTATTGTAGCTAACGCGT60                TAAGCACTCCGCCTCCCCACTCAGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGAC120               CCGCACAAGGCTGGAGCATGTGGTTTAATTCGATGCTACGCGAAAAACCTTACCACTTTT180               TGACATGAAGGTCGTATCCCTCCTAACAGGGGGAGTCAGTTCGGCTGGACCTTACACAGG240               TGCTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG300               CAACCCTCATTCTTAGTTACCAACAGGTAATGCTGGGCACTCTAAGGAAACTGCCAGTGA360               TAAACTGGAGGAAGGTCCCCATGATGTCAAATCAGCACGGCCCTTATAGGGTGGGCTACA420               CACGTGCTACAATGGCAACTACAATAGGTTGCGAGACCGCAAGGTTTAGCTAATCCATAA480               AAGTTGTCTCAGTTCGGATTGTTCTCTGAAACTCGAGAGCATGAAGTCGGAATCGCTAGT540               AATCGTGGATCATCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCA600               CGCCATGGGAATTGGCTTAACTCGAAGCTGGTGTGCTAACCGCAAGGAAGCAGCCATTTA660               AGGTTGGGTTAGTGACTAGGGTG683                                                    (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (oligonucleotide)                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAATTGCTTATAACCTTTTGGTTATAAAT29                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (oligonucleotide)                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TATAGGTACCGTCATTATCTTCCCTAT27                                                 (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (oligonucleotide)                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCATTAGAAATGATGGGTAATACTGTATAA31                                             __________________________________________________________________________

What is claimed is:
 1. A cloned Ehrlichia chaffeensis-specific gene of abiologically pure culture of E. chafffeensis.
 2. The cloned gene ofclaim 1 encoding an Ehrlichia chaffeensis specific antigen.
 3. Thecloned Ehrlichia chaffeensis-specific gene of claim 1 whose gene productspecifically binds an antibody specific for E. chaffeensis.
 4. Anisolated Ehrlichia chaffeensis specific DNA comprising the nucleotidesequence set forth as SEQ ID NO.
 1. 5. An oligonucleotide primerspecific for E. chaffeensis whose sequence is given by SEQ ID NO:
 3. 6.An oligonucleotide primer whose sequence is given by SEQ ID NO:
 4. 7. Anoligonucleotide probe specific for Ehrlichia whose sequence is given bySEQ ID NO:
 5. 8. A diagnostic kit for detecting infection by E.chaffeensis comprising oligonucleotide primers whose sequences are givenby SEQ ID NO: 3 and SEQ ID NO: 4, packaged separately or together, andan oligonucleotide probe whose sequence is given by SEQ ID NO: 5,packaged separately.
 9. An isolated Ehrlichia chaffeensis specific DNAcomprising the nucleotide sequence which is the complement of thesequence of SEQ ID NO.
 1. 10. A method for detecting infection by E.chaffeensis comprising the steps of:providing a sample of blood from apatient suspected of being infected by E. chaffeensis, processing theblood sample to obtain the DNA contained therein, conducting apolymerase chain reaction amplification of the DNA employing a firstprimer specific for E. chaffeensis whose sequence is given by SEQ ID NO:3 and a second primer specific for E. chaffeensis whose sequence isgiven by SEQ ID NO: 4, and probing the resulting amplified sample with aprobe specific for Ehrlichia whose sequence is given by SEQ ID NO: 5,wherein a determination that the probe has bound to the amplified DNAindicates positive detection of infection by E. chaffeensis in the bloodsample.